26th annual meeting of the American Society for Photobiology Non-linear excitation of endogenous and administered fluorophores provides diffraction limited three-dimensional imaging I cells and living tissue preparations. The chief advantages of multiphoton excitation for quantitative imaging are (1) the inherent localization of excitation to a small well-defined volume at the focus of a high numerical aperture objective, and (2) the ability to excite ultraviolet and blue optical transitions with better penetrating near-infrared illumination. The advantages enable high-resolution imaging of fluorescent molecules, including photosensitizing compounds such as Photofrin, within realistic tumor environments. Photofrin excitation is observed to change from a one to two-photon process in the range from 700-740 nm and can be two-photon excited from 750-900 nm. It is characterized by a fluorescence cross section of approximately 0.5 x 10-50 cm4 s/photon throughout this range. Since mitochondrial autofluorescence originating from NADH also undergoes two-photon excitation in this wavelength range, both species can be simultaneously imaged. We observe Photofrin to localize initially to the plasma membrane followed by subcellular localization in mitochondria and possibly other organelles. Even after prolonged incubation, drug is present between cells comprising the tumor spheroids. Drug incubation results in protocol dependent decreases in autofluorescence. Finally, photodynamic action is observed to dramatically alter several aspects of cellular autofluorescence, as well as cell morphology. Supported at DRBIO by NIH RR04224. M.G.N. is a NIH postdoctoral fellow supported by NRSA 1 F32 CA 72225-02.